Affinity Chromatography (AC)
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Affinity Chromatography (AC) 1 Affinity Chromatography (AC) • Principles of AC • Main stages in Chromatography • How to prepare Affinity gel - Ligand Immobilization - Spacer arms – Coupling methods – Coupling tips • Types of AC • Elution Conditions • Binding equilibrium, competitive elution, kinetics • Industrial Examples: Protein A/G for Therapeutic proteins • Future Considerations 2 What is affinity chromatography? Affinity chromatography is a technique of liquid chromatography which separates molecules through biospecific interactions. The molecule to be purified is specifically and reversibly adsorbed to a specific ligand The ligand is immobilized to an insoluble support (“matrix”): resin, “chip”, Elisa plate, membrane, Western, IP (immuneprecipitation), etc Introduction of a “spacer arm” between the ligand and the matrix to improve binding Elution of the bound target molecule a) non specific or b) specific elution method 3 What is it used for? Monoclonal and polyclonal antibodies Fusion proteins Enzymes DNA-binding proteins . ANY protein where we have a binding partner!! 4 Designing and preparing an affinity gel Choosing the matrix Designing the ligand - Spacer arms Coupling methods 5 Ligand Immobilization Ligand + Activating agent + Matrix Activated Immobilised matrix ligand 6 Designing the ligand Essential ligand properties: interacts selectively and reversibly with the target Carries groups which can couple it to the matrix without losing its binding activity Available in a pure form 7 Steric considerations & spacer arms Small ligand (<1,000) Risk of steric interference with binding between matrix and target molecule Often need spacer arm but watch out for Spacer arm adsorption to the spacer! 8 Design of spacer arms Alkyl chain Real risk of unspecific interactions between H H spacer and target molecule O O Hydrophilic chain Risk of unspecific interactions greatly O H reduced No coupling reaction will use 100% of the available binding sites. Using a gel with pre-attached spacer arms will leave some uncoupled spacers, increasing the possibility of non-specific interactions. The unused spacer arms should not be too much of a problem if they are hydrophilic. If available, this problem may be eliminated by using a ligand with a pre-attached spacer arm. 9 Choosing a coupling group • Ligands are coupled to the matrix via reactive groups Amino -NH2 Hydroxyl -OH Aldehyde -CHO Thiol -SH Carboxyl -COOH • Coupling must allow a free binding site 10 Useful Coupling Methods Coupling chemistry Kinds of group coupled Ligand N-hydroxysuccinimide -NH2 Protein ,Peptide. Sugar.Polynucleotide.Cofactor CNBr -NH2 Protein , Sugar.Polynucleotide.Cofactor Carbodiimide -NH2 Small ligands. Non polar ligand in organic via hydrophilic spacer arm solvents. Derivatized material Epoxide -SH-NH2-OH Sugars. Small ligands via hydrophilic spacer arm Thiol exchange -SH Protein ,Peptide. Polynucleotide Mild Conditions Low MW thiol containig ligand 11 Useful Coupling Methods Cyanogen bromide activation O C N O + Gel C N + H Br Gel H Br Sepharose Cyanogen Cyanate ester bromide formed Cyanogen bromide coupling H O Lig NH O N Gel C N + 2 Gel C Lig CNBr-activated NH Protein ligand N-substituted isourea Sepharose Reaction at pH 8. Isourea may be cleaved by nucleophilic attack at high pH. Multiple bonds formed with protein ligands. No spacer arm present. 12 WARNING!: Cyanogen bromide is dangerous. Consult safety data before start working A general protocol for ligand coupling Prepare the ligand solution in coupling buffer Prepare the activated matrix Mix the ligand solution and the activated gel in coupling buffer until coupling is complete Check total ligand before and after coupling: ligand/ml resin Block any remaining active groups Wash the coupled gel alternately at high and low pH to remove adsorbed ligand Transfer to storage buffer 13 Tips: Preparing the activated matrix - couple buffer If the activated matrix is freeze-dried, allow it to swell before washing Always use the recommended swelling and washing solutions! The swelling, washing and equilibration are best performed on a sintered glass filter. Facilitates quickly removal of excess liquid from the media Couple buffer: make sure there are no components, e.g. amines, that can couple in place of the ligand, e.g. Tris buffer has a primary amine To suppress ionic adsorption, add NaCl, 0.5 M. pH is important. Always use the recommended pH. 14 Tips: Coupling reaction, washing and storage Ligands which are poorly soluble in water, may usually be added in an organic solvent. The ligand should be available at a high level of purity. Any contaminants present in the ligand sample will likely be coupled to the matrix. Mix the activated gel and the ligand solution by swirling or end-over-end mixing - Avoid magnetic stirrers as these can crush the gel and produce fines!! Take samples of ligand before and after reaction: to calculate ligand per ml resin Time and temperature according to standard protocols. Most coupling reactions are performed at room temperature Block any remaining active groups with a small neutral ligand (Tris buffer for amino reactions) Wash the coupled gel at alternating high and low pH to remove adsorbed substances. 15 Keep in storage buffer (Na Azide , Thimerosol 0.02% or ethanol if possible) G Protein Receptor Kinase Technique Pig brain homogenate Purification Comment factor Ppt Ammonium sulfate 7 precipitation A. Tobin et al. (1996) J. Biol. Chem. 271, 3907-3916 HIC Butyl Sepharose Fast 20 Flow AIEX RESOURCE Q CIEX RESOURCE S 2408 AC HiTrap Heparin 18647 • 10 mg homogenous protein 16 Membrane Protein Technique Placental extract in Purification Comment 1.5% Triton X-100 factor AC Blue Sepharose 3 • Concentration and capture AIEX DEAE Sephacel 4 T. White et al. (1995) J. Biol. Chem. 270, 24156-24165 CIEX SP Sepharose 6 AC Muc2 Sepharose 242 • Main purification step • Final polishing and purity CIEX Mono S 1442 check, 20 mg obtained 17 DNA-binding Protein Technique HeLa cell nuclear Purification Comment extract factor J. Berthelsen et al. (1996) J. Biol. Chem. 271, 3822-3830 CIEX SP Sepharose High 5 Performance AC Heparin Sepharose 8 • General AC step for DNA- Fast Flow binding proteins AC DNA-1 Sepharose 9 • Removal step, non-specific DNA binding activity removed AC DNA-2 Sepharose 2447 • Main purification step CIEX Mono S 4943 • Final polishing, 20 mg obtained 18 Advantages and Disadvantages of affinity chromatography • Easy to achieve otherwise difficult • Buffer / elution limitations: Sometimes needs harsh or denaturizing separations elution conditions Very expensive elution conditions • Often high purity in one step (but not always) • Economics (scale-up): Ligand stability affects life-time of • Relatively fast separations affinity resins (according to kinetics of binding and elution) Some ligands are very expensive • Can be use to remove specific • Do not resolve all the problems contaminants 19 The main stages in affinity chromatography Sample application Elution of the target Equilibration Wash Re-equilibration Of gel and sample contaminants Of the gel to binding conditions to binding conditions Or keep in storage buffer (20% Ethanol or 0.02%NaAzide in Buffer) 20 Columns and equipment Tips Column volume: according to amount of target and gel capacity Avoid using excess of resin: to avoid low affinity binding of impurities. Use maximum capacity of the column. Column length: not usually critical, short and wide Equipment: no special demands Equilibrate column before applying sample and follow standard protocol 21 Sample preparation Tips Adjust pH, buffer salts and additives to promote binding Filter or centrifuge to remove particles Make sure that components known to interfere with binding are absent Consider alternative capture step before affinity purification Eliminates crude impurities or interfering substances IEX or HIC columns are cheaper, with higher dynamic capacity and quickly concentrate lysates Prolongs resin half life (reduce cost) 22 Sample application Tips Binding buffer: usually neutral pH, and high salt concentration when is possible (0.3-0.5M NaCl) to avoid hydrophobic interactions of non specific proteins to the resin. Use additives only if necessary. Loading directly on column - Strong affinity and fast binding: High flow rate - Weak affinity and/or slow binding: Low flow rate 23 Kinetics of adsorption and desorption Unexpected results Slow binding Some targets do not completely bind to the resin: very low dynamic binding capacity Open columns: slow down binding. Stopped flow binding to bind the target in portions or rebind several times For batch binding, incubation of around 1.5 hours at 4°C is enough. For extremely slow binding kinetics, incubate overnight at 4°C. Very gentle stirring Binding on FPLC column: adjust flow rate to get loading in ~90min Slow desorption If target elutes as a long, low 'peak‘, reduce elution flow-rate Open columns: Stopped flow elution to elute the target in pulses. Change elution scheme Elution of FPLC column: extremely slow elution in order to get narrow peaks 24 Washing, elution and re equilibration Tips • Follow standard protocol • Optimize alternative wash strategies to increase final purity (high salt, detergents, others) • If eluting at extreme pH: collect the target protein in a small amount of concentrated buffer at a neutralizing pH • Re-equilibrate column immediately with binding or storage buffer • Regeneration: use buffers that do not harm the ligand 25 Column Regeneration Tips Wash with basic and acidic buffers. – 10 col. vol. 0.1 M Tris.HCl, 0.5 M NaCl, pH 8.5 – 10 col. vol. 0.1 M NaAc, 0.5 M NaCl, pH 4.5 – Use chaotropic agents or detergents if needed Re-equilibrate with 10 column volumes starting buffer. Use more if needed Ni columns can be washed with 0.5N NaOH, neutralized, destriped with 100mM neutral EDTA, wash, charge with 100mM Ni SO4, wash and store with 20% Ethanol 26 Column Storage Tips To maximize the useful lifetime of an affinity resin, choose a storage buffer that does not harm the ligand Typical buffers are 20% ethanol solution or binding buffer containing a bacteriostatic agent (e.g.